11 research outputs found

    Ab417 binds to L1CAM-expressing ICC cells and reduces L1CAM levels at the membrane.

    No full text
    <p>(A) Confocal microscopic analysis of Ab417 internalization. SCK-L1 cells were preincubated with Ab417 (10 μg/mL) for the indicated times. Membrane L1CAM-bound Ab417 was detected using anti-Human IgG(H+L) Cross Adsorbed DyLight 594 (red), and internalized Ab417 was detected using anti-human IgG (Fc-specific)-FITC (green). (B) Choi-CK cells were incubated with Ab417 (10 μg/mL) for the indicated times, and the membrane L1CAM was detected by western blot analysis using A10-A3 and pan-cadherin as a loading control. Control (c) indicates the cells that were incubated for 6 h without Ab417. (C) Confocal microscopic images of L1CAM staining of PBS (up)- or Ab417 (down)-treated Choi-CK tumors. The images were taken at x400 magnification. L1CAM index (D) are represented as a percentage of positively-stained area comparing to total area.</p

    Anti-tumor efficacy of gemcitabine or cisplatin in a Choi-CK xenograft model.

    No full text
    <p>Drug at indicated dose was <i>i</i>.<i>p</i>. injected twice a week for 3 or 4 weeks, and tumor volume (A) and tumor weight (B) were determined. Each point indicates the mean ± s.d. <i>p</i> < 0.05 (*) and <i>p</i> < 0.01 (**), significant difference from the saline-treated group by Dunnett’s <i>t</i>-test. <i>p</i> < 0.05 (<sup>#</sup>) and <i>p</i> < 0.01 (<sup>##</sup>), significant difference from the saline group by Steel’s test.</p

    Anti-tumor efficacy of combined treatment with Ab417 and gemcitabine or cisplatin in a Choi-CK model.

    No full text
    <p>Tumor volume (A and C) and tumor weight (B and D) are represented. Each point indicates the mean ± s.d (<i>n</i> = 8). <i>p</i> < 0.05 (*) and <i>p</i> < 0.01 (**), significant difference from the saline group by Dunnett’s <i>t</i>-test. <i>p</i> < 0.05 (<sup>#</sup>) and <i>p</i> < 0.01 (<sup>##</sup>), significant difference from the saline group by Steel’s test.</p

    Anti-tumor efficacy of Ab417 in a Choi-CK xenograft model.

    No full text
    <p>Ab417 (10 mg/kg) or the isotype control (hFc, 3.3 mg/kg) was <i>i</i>.<i>v</i>. injected twice a week into nude mice bearing Choi-CK or TFK-1 xenografts (<i>n</i> = 8). Tumor volume (A), tumor weight (B), and body weight (C) were represented. Each point indicates the mean ± s.d. <i>p</i> < 0.05 (*) and <i>p</i> < 0.01 (**), significant difference from the isotype control group by Dunnett’s <i>t</i>-test. <i>p</i> < 0.05 (<sup>#</sup>), significant difference from the isotype control group by Steel’s test. (D) Tumor sections were stained with an anti-human Ki-67 antibody. Ki-67 index are represented as a percentage of positively-stained area comparing to total area.</p

    Secondary structures in 5’UTR correlate with translational repression of target genes.

    No full text
    <p>(<b>A</b>) A hairpin structure in the <i>CD69</i> 5’UTR. (<b>B</b>) Ribosome accumulation in <i>CD69</i> 5’UTR correlated with miR-17~92 family miRNA expression levels. Note that the hairpin structure co-localizes with the ribosome footprint peak in the <i>CD69</i> 5’UTR. <i>Actb</i> was used as control. (<b>C</b>) Deletion of the miR-17~92 family miRNAs shifted <i>CD69</i> mRNA from light to heavy polysomes. (<b>D</b>) Increased <i>CD69</i> expression in TKO B cells was mainly due to translation de-repression. Experiments in <b>B-D</b> were performed with 25.5h activated B cells.</p

    Quantification of miR-17~92 miRNAs and binding sites in primary B cells.

    No full text
    <p>(<b>A,B</b>) Quantitative Northern blot to determine miR-17~92 miRNA copy numbers. Indicated amounts of synthetic miR-17, miR-18a, miR-19b and miR-92 were added to naïve and activated TKO B cells before RNA extraction. The copy numbers of each miRNA subfamily were determined by Northern blot comparing WT B cells and TKO B cells with graded amounts of spike-in synthetic miRNAs, using a mixture of probes corresponding to all members of a miRNA subfamily (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s029" target="_blank">S7 Table</a></b>). Naïve B cells were activated with LPS and IL-4 for indicated amounts of time (h, hour). (<b>C-E</b>) Summary of miR-17~92 family miRNA copy numbers (<b>C</b>), conserved miR-17~92 family miRNA binding sites (<b>D</b>) (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s030" target="_blank">S8 Table</a></b>), and ratios of conserved miR-17~92 family miRNA binding sites to miRNAs (<b>E</b>) in naïve and activated B cells.</p

    Target genes exhibit different sensitivity to miR-17~92 expression level changes.

    No full text
    <p><b>(A)</b> Minimal overlap between ribo-upregulated TKO targets and ribo-downregulated TG targets. <b>(B-D)</b> The responses of ribosome density of ribo-upregulated TKO targets <b>(B)</b> and ribo-downregulated TG targets <b>(C)</b> to three miR-17~92 expression levels. Translated genes lacking miR-17~92 binding sites were used as control <b>(D)</b>. Colored bars indicate median values and error bars represent interquartile ranges. Each dot represents relative ribosome density of a unique gene. Numbers indicate p-values. <b>(E)</b> Different sensitivity of individual target genes to miR-17~92 expression level changes. Protein levels were determined by immunoblot and normalized to β-Actin (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s011" target="_blank">S11</a> Figs</b>). Target gene protein levels in WT B cells were arbitrarily set as 1.0 (n≥4). Vertical lines indicate error bars. <b>(F)</b> Relative mRNA levels of individual target genes in TKO, WT and TG B cells as determined by microarray (n = 3). <b>(G)</b> A hypothetical curve depicting target gene protein level change as a function of miRNA concentration. For a miRNA-target mRNA interaction in a given cellular context, there are a threshold level and a saturation level of miRNA concentration. miRNA suppresses target gene expression in a dose-dependent manner when miRNA concentration is between the threshold and saturation levels. Suppression does not occur when miRNA concentration is below the threshold level, while suppression reaches a maximal when miRNA concentration is above the saturation level. <b>(H)</b> The hypothetical response curves of group1, group2 and group3 target genes to miR-17~92 expression level changes. Note that the difference in amplitude for individual target genes is not depicted in this graph.</p

    Ribosome accumulation in 5’UTR correlates with translational repression of target genes.

    No full text
    <p>(<b>A-C</b>) Ribosome accumulation in 5’UTRs of ribo-upregulated TKO targets in WT B cells (<b>A</b>), ribo-downregulated TG targets in TG B cells (<b>B</b>), but not in 5’UTRs of other targets (<b>C</b>). Ribosome occupancy in 5’UTR was normalized to the overall ribosome footprint abundance of the same gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.ref096" target="_blank">96</a>]. The first nucleotide of start codon is set as position 0 (grey dashed line). (<b>D</b>) Inverse correlation between ribosome occupancy in 5’UTR and the overall ribosome density on target mRNA in WT B cells. (<b>E</b>) High GC content in 5’UTRs of ribo-upregulated TKO targets.</p

    Regulation of target gene sensitivity to miRNA suppression by 5’UTR.

    No full text
    <p>(<b>A</b>) An engineered psiCheck2 vector (psiCheck-2-pd) for investigating the effect of 5’UTR and 3’UTR on reporter gene expression. TSS, transcription start site. (<b>B</b>) Experimental scheme of reporter assays in primary B cells. FACS plots show electroporation efficiency using a GFP-expressing plasmid. (<b>C,D</b>) Dual luciferase reporter assay to determine the effect of 5’UTR and 3’UTR on the reporter gene protein (luciferase activity) (<b>C</b>) and mRNA (qRT-PCR) levels (<b>D</b>). Closed and open circles indicate reporters with wild-type (wt) and mutated (mut) <i>CD69</i> 3’UTR, respectively. A comparison of renilla luciferase activity normalized to firefly luciferase activity (hRluc/Fluc) between psiCheck-2-pd containing mut and wt <i>CD69</i> 3’UTR reveals the sensitivity of the renilla luciferase mRNA (hRluc) to miR-17~92-mediated suppression. Results of normalized hRlcu/Fluc (n = 10) are from three independent experiments. Each experiment contained 3–4 replicates.</p

    The impact of miR-17~92 on target gene mRNA and protein levels.

    No full text
    <p>(<b>A</b>) The distribution of mRNA abundance in naïve and activated B cells as determined by ERCC-RNA-seq analysis. Numbers in parenthesis represent the number of unique genes significantly transcribed (greater than 0.5 copy per cell). Y-axis (counts) indicates the number of genes of a given abundance (X-axis, bin size = 0.2). (<b>B,C</b>) Microarray analysis of TKO, WT, and TG B cells. Numbers in parenthesis indicate the numbers of transcribed genes and transcribed miR-17~92 targets analyzed by microarray. (<b>D</b>) The protein and mRNA levels of 13 target genes showing reduced protein levels in 25.5h activated TG B cells as determined by Immunoblot (n = 5). mRNA levels were determined by qRT-PCR and microarray (n = 3). Target gene expression levels were normalized to β-Actin, and their relative expression in WT naïve B cells was arbitrarily set as 1.0.</p
    corecore